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Nutrition Research Center, Department of Physiology, St. John's Medical College, Bangalore 560034, India;
*
U.S. Department of Agriculture/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX;
Department of Pediatrics, St. John's Medical College, Bangalore, India; and
**
U.S. Department of Agriculture, Washington, DC
3To whom correspondence and reprint requests should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoisocaproic acid. The prevaccination
oxidation of leucine (percentage of dose ± SD) was
18.1 ± 2.3 (men) and 16.7 ± 3.8 (children). One day after
vaccination, these values had risen to 19.9 ± 1.9
(P < 0.05) in the men and to 19.5 ± 4.6
(P < 0.01) in the children. In the adults,
vaccination was associated with a rise in whole-body protein
breakdown [mg protein/(kg·h)] from 200 ± 40 to 240 ± 10
(P < 0.05). A minor simulated infection increases
leucine catabolism in undernourished humans and this new, minimally
invasive protocol is sufficiently sensitive to measure these
changes.
KEY WORDS: • infection • protein metabolism • undernourished humans • stable isotopes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Waterlow 1973
, Wells et al. 1993
, WHO 1986
). This reduced growth rate, which
leads to stunting in later life, is associated with long-term
effects, including decreased productivity and functional deficits
(Spurr 1988
, Vaz et al. 1996
). The cause
of this reduction in growth rate is likely multifactorial, reflecting
the interactions of a poor diet and a poor environment (Henry 1981
) and, in particular, the consequences of bacterial
infections and parasitic infestations (Scrimshaw et al. 1959
).
In a recent paper on underprivileged children in developing countries,
Solomons et al. (1993)
proposed that the occurrence of
"inapparent" infections in children (no clinical findings, but
abnormal erythrocyte sedimentation and white cell count) is a factor
that increases the demand for nutrients. They argued that during
chronic immunostimulation, there is a partitioning of nutrients toward
the support of the immune defenses and an effective reduction in the
availability of nutrients for growth.
Among other factors, the immune response involves a
cytokine-stimulated (Jennings et al. 1992
,
Keusch 1982
, Kushner 1982
) increase in
the synthesis of so-called positive acute-phase proteins by the
liver. This increased liver protein synthesis has to be supported
either by amino acids derived from the diet or by breakdown of body
protein. However, it is clear that there is a cost to this process
because even when there is an apparently adequate supply of dietary
protein, there is still a net loss of nitrogen from the body during
active infection Reeds et al. (1994)
. The loss of body
protein after the stress of injury or infection is largely from
skeletal muscle (Essen et al. 1993
, Souba et al. 1990
). It has been estimated that the synthesis of acute phase
proteins can peak at 1.2 g/(kg body weight·d) (Waterlow 1991
). Reeds et al. (1994)
hypothesized that
negative nitrogen balance occurs during an acute-phase response
because the amino acid composition of the positive acute phase proteins
differs from that of muscle protein (Barker 1984 and 1987
). This leads to an internal amino acid imbalance, and as a
result, 2 g of mixed muscle protein must be broken down to support
the synthesis of 1 g of the typical mixture of positive acute
phase proteins. The excess amino acids liberated from muscle are then
oxidized, leading to an increased N loss from the body.
The quantitative nutritional effect of infection has proved difficult
to assess. This paper reports the application of an isotopic protocol
that attempts to provide such information. We investigated the utility
of a minimally invasive approach (Fjeld 1994
) to
determine leucine oxidation in young children. The method was of short
duration and involved only the administration of an oral tracer and the
collection of breath. We also used the approach in an analogous study
in adults and compared the results with those obtained using the more
conventional, but invasive method of blood sampling. As part of the
investigation, we used the noninvasive protocol in both adults and
children to quantify the changes in leucine oxidation associated with
vaccination.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study 1
Subjects.
Five clinically healthy, but chronically undernourished (by the
criteria defined in James et al. 1988
, i.e.,body mass
index < 18.5 kg/m2,as well as low socioeconomic
status) adult male subjects aged 18–26 y were recruited from a
neighboring urban slum. They lived in temporary dwellings, which had
neither sanitation nor water supply. The socioeconomic status of the
subjects was assessed by an education, occupation and family income
scale (Kuppusawamy 1984
). A clinical examination along
with routine blood and urine analyses was carried out before commencing
the experimental protocol.
Anthropometric measurements.
Anthropometric and skinfold measurements were made on d 1, at the start
of the study. Duplicate measurements of the subjects' weight, to the
nearest 0.1 kg, were made using a digital scale (Soehnle GmBH, Hamburg,
Germany) with the subject dressed in minimal clothing. The heights of
the subjects were recorded without footwear, using a vertically mobile
scale (Karrimetre, Uppsala, Sweden) and expressed to the nearest 0.1
cm. Duplicate measurements of the biceps, triceps, subscapular and
suprailiac skinfolds were made with the subject in the standing
position. All skinfold measurements were standardized (Harrison et al. 1988
) and carried out to the nearest 0.2 mm, using
skinfold calipers (Holtain, Crymmych, UK). The logarithm of the sum of
the four skinfolds was used, in age- and gender-specific equations
(Durnin and Womersley 1974
) to obtain an estimate of
body density, from which percentage body fat was determined
(Siri 1961
).
General protocol.
The study lasted for 10 d. On d 1, the subjects were brought in
for clinical examination and anthropometric measurement. For the next
2 d (adaptation period), the subjects consumed a standardized
diet, and on d 3, they were studied with the tracer protocol (see
below). They were then allowed to go home. The subjects returned to the
laboratory on d 7 for a further 2 d of controlled feeding. On d 9,
they were vaccinated with the
DPT4
(diphtheria, pertussis, and tetanus; Biological Ltd., Hyderabad, India)
vaccine intramuscularly into the deltoid muscle. The vaccine contained
diphtheria and tetanus toxoids, and killed whole-cell pertussis
bacteria, with aluminum phosphate as adjuvant. A blood sample was
collected 24 h after the vaccination. The DPT vaccine was
administered as a 0.5-mL intramuscular injection with no side effects.
On d 10, the subjects were once again studied with the tracer protocol
(Fig. 1
).
|
).
The diet consisted of rice, lentils, vegetable oil and vegetables. On
the basis of food tables (Gopalan et al. 1996
,
McCance and Widdowson 1995
), the diet contained 75%
carbohydrate, 12% fat and 13% protein and supplied 88 mg (0.67 mmol)
leucine/(kg · d).
Tracer protocol.
The tracer protocol was carried out on two occasions,i.e, on d 3 (6 d
before vaccination) and on d 10 (1 d after the administration of the
vaccine). On the day of the tracer study, the subjects were awakened at
0600 h. Samples of urine and feces were collected and sent for
routine microscopic and biochemical tests. The subjects then consumed
frequent meals of a diet of semolina porridge for the 6-h duration of
the protocol. The intake over 6 h provided one quarter of their
estimated daily energy requirements in an inactive state (1.3 x BMR) (FAO/WHO/UNU 1985
)and was given at half-hour
intervals (see Fig. 2
). The meal was always given after the tracer dose. The diet supplied 61
mg leucine/(kg · d), thereby ensuring that the leucine intake from
diet and tracer was the same as the daily leucine intake during the
adaptation period.
|
. After the
priming dose, intermittent doses of NaH13CO3
[0.166 mg/(kg · 20 min)] were given every 20 min for a period of
3 h. Breath samples were taken every 20 min starting just before
administration of the isotope. After 3 h, the
NaH13CO3 was stopped and a priming dose of
13C leucine (99.9%, Cambridge Isotopes) (2 mg/kg) was
given, followed by intermittent hourly doses of 13C leucine
[2 mg/(kg · h)] every hour for the next 3 h. Breath samples
continued to be taken at 20-min intervals and blood samples were
collected every 20 min during the last hour of the experiment. Indirect
calorimetry (Shetty et al. 1987
) was carried out on all
subjects during the 6 h of the protocol in order to measure the
excretion rate of CO2(VCO2).
Urine was collected in urine bags every 3 h for the next 24 h. Each void was acidified with a known volume of acid, measured and
stored in separate containers for subsequent analysis of total urinary
N (Varley 1980
). In addition, a blood sample was
collected before the start of the protocol and at the end of the 24-h
urine collection period to measure plasma urea concentration in order
to correct for the effect of the change in the urea pool size on N
excretion rate (Kaplan 1965
).
Study 2
Subjects.
Nine children (4 boys and 5 girls, mean age 4.1 ± 0.6 y)
were studied. They appeared healthy by clinical examination but were
stunted when viewed against the NCHS standards (National Center for Health Statistics 1990
). They lived in slums in poor hygienic
conditions and had neither water nor sanitation in their homes. Their
parents were of very low socioeconomic background. Information on their
past medical and nutritional histories was obtained by questionnaire,
and their family socioeconomic status was measured by the
Kuppuswamy (1984)
questionnaire.
Anthropometry. The anthropometric measurements were performed as described for adults.
Adaptation.
For 2 d before the isotopic study, the children were fed according
to their requirement (WHO/FAO/UNU 1985
) [~370 kJ/(kg
· d)] a diet of rice, lentils, vegetable oil and vegetables. On the
basis of food composition tables (Gopalan et al. 1996
,
McCance and Widdowson 1995
), the macronutrient nutrient
composition of the diets was carbohydrate 75%, fat 12% and protein
13%. The diet supplied 180 mg (1.37 mmol) leucine/(kg body weight ·
d).
General protocol. The general protocol followed was exactly similar to the adult protocol described above.
Tracer protocol.
The protocol administered to the children pre- and postvaccination was
similar in general procedure to the adult protocol; the main exception
was that no blood samples were taken. The children were admitted to the
Metabolic Unit at 0700 h and acclimated to the laboratory for
2 h. Based on their maintenance requirements (209 kJ/kg), the
children were then fed a diet of wheat starch (protein-free) biscuits.
These were made into a mush with water and given as small divided meals
every half-hour during the 6-h protocol (see Fig. 2
). Each meal was
given after the tracer dose.
After a basal breath sample was collected, a priming dose of
NaH13CO3 (0.5 mg/kg) was given orally
followed by intermittent maintenance doses of
NaH13CO3 (0.166 mg/kg) given every 20 min for a
period of 3 h (Fig. 2)
. Breath samples were also taken every 20
min starting just before administration of the isotope. After 3 h,
the NaH13CO3 was stopped and a priming dose
of 13C leucine (3mg/kg) was given, followed by intermittent
maintenance doses of 13C leucine (3mg/kg) every hour for
the next 3 h. Breath samples were collected at 20-min intervals.
The analysis, calculations and statistics are common to Study 1 and 2.
Laboratory analysis. All clinical laboratory analyses were done by routine procedures at the St. John's Medical College Hospital Central Laboratory. Urine and fecal samples were sent for routine microscopic and biochemical tests.
The breath samples were analyzed in duplicate with an isotope ratio
mass spectrometer (Europa Scientific, Crewe, UK) by the continuous flow
technique. The precision of this technique was ± 0.001 atom %
(SD). Plasma -keto isocaproic acid (KICA) isotope
ratio was determined with a Hewlett Packard (Palo Alto, CA) 9890
quadrupole mass spectrometer by negative chemical ionization gas
chromatography/mass spectrometry of the pentafluorobenzyl derivative,
monitoring ions at m/z 129 and 130.
Calculations.
Percentage of 13C leucine oxidized (R)
 | (1) |
where breath 13CO2 enrichment (atom % excess) was measured during the last hour of 13C leucine administration.
 | (2) |
Leucine flux (Q) [µmol/(kg · h)]
 | (3) |
in which MPE is the tracer:tracee ratio of 13CKIC
(mol % excess). Leucine oxidation (E)[µmol/(kg · h)]
 | (4) |
 | (5) |
 | (6) |
Protein synthesis (by tracer method) [mg protein/(kg · h)]
 | (7) |
in which 131.17 is the molecular weight of leucine and 0.08 is the leucine content of body protein (g/kg).
Protein breakdown (by tracer method) [g protein/(kg · h)]
 | (8) |
 | (9) |
Corrected urinary N excretion [mg/(kg · h)]
 | (10) |
where TBW is total body water estimated after Siri (1961)
, and 0.92 is a correction term for urea concentration in
plasma water.
Corrected equivalent protein excretion [g
protein/(kg · h)]
 | (11) |
Total amino-N flux [mg N/(kg · h)]
 | (12) |
with the rate of excretion of 13C in
breath CO2 from .
 | (13) |
Statistical analysis. Because of the small sample size, differences were tested with nonparametric tests (Wilcoxon) for paired comparisons and were considered significant if P < 0.05. Correlation was performed by Pearson's product-moment method. Results are expressed as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preliminary studies to determine the contribution of the diet to the 13C enrichment of CO2 in the breath showed a negligible change in the isotopic enrichment of expired carbon dioxide (<0.001 atom % excess) after feeding.
Study 1.
The subjects' mean height was 164.7 ± 4.1 cm, their weight was
44.0 ± 2.8 kg and their body mass index was 16.3 ± 0.9
kg/m2. There was no change in their weight during
the 10 d of the study. Two of the subjects had Escherichia
coli in their fecal samples, but all other routine laboratory
tests were normal. After the vaccination, on the study morning, two of
the subjects had a mild pyrexia (39°C), and all complained of pain
and stiffness at the injection site. The hematological measurements
(Table 1) showed that vaccination caused a significant increase in total count
of white blood cells (WBC) and neutrophils (P < 0.05),
but no change in hemoglobin. The erythrocyte sedimentation rate (ESR)
was not available after vaccination, due to technical error.
|
). The between-sample coefficient of variation of the
13CO2 isotopic enrichment
at plateau was ~5% during the infusion of either labeled bicarbonate
or leucine. Table 2
shows the individual data for plasma KIC labeling, leucine oxidation
and leucine kinetics. The isotopic enrichment of plasma KIC was
significantly (P < 0.05) lower and the percentage of
dose oxidized and leucine flux were significantly (P < 0.05) higher during the second (postvaccination) study. The increase in
oxidation after vaccination was not associated with an increase in the
RA of CO2 (Table 3). There was no significant difference in the plasma urea between the
start and the end of the protocol either before (8.6 ± 2.6 vs.
8.2 ± 2.9 mmol/L plasma) or after the vaccination (7.0 ± 3.2 vs. 7.8 ± 3.2 mmol/L plasma).
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, the results for leucine metabolism from the invasive (tracer method)
and the noninvasive breath/urine based method are compared. The rates
of protein synthesis calculated by the two methods were not
significantly different. In neither case did vaccination have a
significant effect on whole-body protein synthesis, although
whole-body protein breakdown, calculated by the tracer method, was
significantly higher 24 h after vaccination than 7 d before.
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).
|
The mean age, weight and height of the children were 4.1 ± 0.6 y, 12.3 ± 1.3 kg, and 92.5 ± 3.2 cm, respectively.
Their mean mid-upper arm circumference was 14.3 ± 1.0 cm; the
mean head and chest circumferences were 48.1 ± 1.5 and 49.7
± 2.3 cm, respectively. The children were malnourished, with
Z-scores (National Center for Health Satistics 1990
) of -2.4
± 0.9 for height-for-age; -2.3 ± 0.7 for weight-for-age;
and -1.2 ± 0.8 for weight-for-height. The clinical examination
was normal, and routine urine and fecal examination showed no
abnormality. Their hemoglobin level was 119 ± 10 g/L, the total
WBC count was 9.9 ± 3.8 x 109
cells/L, with neutrophils 44.4 ± 17.5%, lymphocytes 46.3 ± 17.6% and eosinophils 6.6 ± 2.6%. The ESR was 20.0 ± 16.0
mm/h. The high SD of the latter was due to one subject who
had an ESR of 56 mm/h. There was a mild pyrexia (+1°C) in five of the
children after vaccination. This was controlled, for ethical reasons,
with 120 mg of paracetamol. All of the subjects complained of stiffness
and pain at the site of the injection, along with decreased mobility.
Apparent steady states in the enrichment of CO2
were seen after 2 h of 13C-bicarbonate
administration (Fig. 4
). The calculated RA of CO2
in both phases is presented in Table 3
. There was a significant
(P < 0.01; 17%) increase in the oxidation of
13C-leucine, but no significant change in the
RA of CO2 after
vaccination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Under the stimulus of
proinflammatory cytokines such as interleukins (Dinarello 1984
), endocrine changes that lead to the mobilization of amino
acids from the periphery, primarily from skeletal muscle (Essen et al. 1993
, Souba et al. 1990
), are initiated.
The amino acids so released are taken up in the splanchnic tissues
(Souba et al. 1990
) and used for the synthesis of the
positive acute phase proteins (Colley et al. 1983
). It
has been argued (Reeds et al. 1994
) that the associated
loss of body protein reflects an amino acid imbalance that results from
the dissimilarity of the amino acid composition of the acute phase
proteins [high in aromatics and cysteine, low in branched-chain
amino acids (BCAA)] and muscle protein (low in aromatics and cysteine,
high in BCAA). According to this hypothesis, amino acids, notably the
BCAA, that are mobilized in excess of the need for acute phase protein
synthesis are "wasted" via excess oxidation. The primary
objective of this study was to quantify whether this occurs during an
"infection" as simulated by vaccination. A secondary objective
was to use the investigation to examine the utility of a minimally
invasive isotopic protocol that might be applicable to larger-scale
studies in the field. Methodological considerations.
In designing the isotopic approach to test our main hypothesis, i.e., that infection is associated with an increased oxidation of leucine, a number of points have to be considered.
First, although studies performed in the postabsorptive or fasting
state simplify the interpretation of the isotopic data, the main
nutritional issue is whether concurrent infection lowers the ability of
the individual to retain dietary amino acids. We therefore performed
the studies in fed subjects. This poses some limitations. First,
because leucine oxidation is closely related to leucine intake in the
diet, (El Khoury et al. 1995a and 1995b
,
Pelletier et al. 1991
), the study should be performed at
a relatively low but nutritionally adequate leucine (protein) intake.
Second, to infer changes in protein balance from the metabolism of a
single tracer amino acid, it is important to use diets in which the
contribution of the test amino acid is close to its contribution to
body protein.
In addition, we also wished to develop a protocol that involved as
little manipulation of the subject as possible and specifically one
that did not rely on data from the labeling of circulating leucine or
its metabolites. Thus, we used oral tracers and based our main
conclusions on data obtained only from the analysis of urine and
breath. In the adult study, we were able to compare the results from
the noninvasive tracer protocol against those from a more conventional
protocol that involved blood sampling and analysis of the labeling of
circulating -keto isocaproic acid (Matthews et al. 1982
). The results from the two approaches were very similar.
However, although the breath/urine "end product" method has the
theoretical advantage of not involving any assumptions with regard to
precursor amino acid labeling, the variability of the values for
protein synthesis derived from the end product approach was high. This
highlights the main limitation to the approach, i.e., the wide
variability of urinary N excretion when it is measured over short
periods of time. Although the method has been used successfully in
animals (Yagi and Walser 1990
) and in humans (El Khoury et al. 1995b
) under well-controlled conditions, the
quantification of true body N loss from urine, as an index of net
protein oxidation (Munro 1964
), is neither easy nor
accurate under field conditions.
An important advantage of the short, noninvasive protocol is that there is no need for indirect calorimetry. Indeed, it can be argued that because the method that we used measures CO2 production (RA CO2), it is a more theoretically appropriate approach to the isotopic study of substrate oxidation, and its use obviates the need to measure or assume a recovery factor for excreted CO2.
Response to vaccination.
The main objective of this study was to investigate the changes in
leucine metabolism after an experimental or simulated infection.
Previous studies (Garlick et al. 1980
), using
15 N glycine, found an increase in whole-body
protein synthesis of 37% ~12 h after vaccination. This is similar to
other reports of increased protein synthesis after severe infection
(Long et al. 1977
) in humans, and during
turpentine-induced stress in rats (Wusteman et al. 1990
). In this study, the protein synthesis rate in the men
increased by only 15%. However, there was a proportionally greater
increase in protein breakdown, and protein balance became more
negative. In both studies, vaccination increased leucine oxidation, and
the increase in the children was higher than that in the adults. This
suggests that the metabolic stress of infection may be greater in
children. The administration of paracetomol could have also mitigated
this response, although paracetomol acts centrally to inhibit
prostaglandin synthesis (Dascombe 1985
, Vane 1971
), with little peripheral anti-inflammatory effect. The
higher response in children may also reflect a booster effect of other
recent immunizations.
In conclusion, this 13C leucine tracer protocol appears to be sufficiently sensitive to detect protein metabolic changes imposed by the relatively minor stress of vaccination. For comparisons that do not require estimates of protein synthesis rates, the protocol requires only a breath collection and attention to the constancy of prior leucine intake. Estimates of protein synthesis can be obtained by the addition of a complete urine collection, plus two blood samples for correction of changes in urea pool size. We believe that this protocol will prove useful in metabolic studies performed outside a metabolic unit setting.
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FOOTNOTES |
|---|
2 The contents of this publication do not
necessarily reflect the views or policies of either the U.S. Department
of Agriculture or the IAEA. Mention of trade names, commercial
products, or organizations does not imply endorsement from the U.S.
government.
4 Abbreviations used: BMR, basal metabolic rate;
DPT, diphtheria, pertussis and tetanus vaccination; ESR, erythrocyte
sedimentation rate; KIC, -ketoisocaproic acid; MPE, moles percent
excess; RA, rate of appearance; TBW, total body water; WBC,
white blood cells.
Manuscript received February 18, 1999. Initial review completed March 10, 1999. Revision accepted April 16, 1999.
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